Increasing vancomycin susceptibility in vancomycin resistant enterococci by vanH promoter and ddl transformation

Abstract

OBJECTIVES: In vancomycin resistant enterococci (VRE) the vanHAXYZ genes encode a new pathway of enzymes to produce d-alanyl-d-lactate. We investigated the effect of vanH promoter and ddl gene transformation on vancomycin susceptibility in a vanA phenotype of Enterococcus faecalis. METHODS: To construct plasmid pJW1, the vanH promoter was cloned to plasmid pAM401. Plasmid pJW2 was constructed by cloning the ddl gene into pAM401. To construct pJW3, the ddl gene was ligated downstream of the vanH promoter of the plasmid pJW1. The competent VRE was transformed with pJW1, pJW2 and pJW3 using electroporation. The minimal inhibitory concentration (MIC) of vancomycin for VRE and transformed E. faecalis was determined using the broth dilution method. The expression of the vanA and ddl gene of VRE and transformed E. faecalis was evaluated by RT PCR. RESULTS: The transformation of the vanH promoter reduced the vancomycin MIC of VRE. In VRE and transformed E. faecalis, the vanA and ddl genes were expressed. CONCLUSIONS: This study presents a way of altering high-level vancomycin resistance with gene transformation in enterococci. In the future, development of an effective gene delivery system will contribute to the design of new modalities that will help overcome the limitations of antimicrobial therapy.